Methods of treating dna damage

ABSTRACT

The present invention relates to methods and compositions of treating patients suffering from, or at risk for, DNA damage and to increase life span, i.e., prevent or slow the aging process in all species. The treatment includes administering to the patient a pharmaceutical composition that includes carbon monoxide.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.14/236,756, filed on Apr. 2, 2014, which is a U.S. National PhaseApplication under 35 U.S.C. § 371 of International Patent ApplicationNo. PCT/US2012/049961, filed on Aug. 8, 2012, which claims the benefitof U.S. Provisional Patent Application Ser. No. 61/521,566, filed onAug. 9, 2011, the entire contents of which are hereby incorporated byreference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. Government support under grant numbersNIH R01GM088666-01 and AHA 10SDG2640091. The Government has certainrights in this invention.

TECHNICAL FIELD

This invention relates to the treatment of DNA damage and/orfacilitation of DNA repair.

BACKGROUND

Heme oxygenase-1 (HO-1) catalyzes the first step in the degradation ofheme. HO-1 cleaves the α-meso carbon bridge of b-type heme molecules byoxidation to yield equimolar quantities of biliverdin IXa, carbonmonoxide (CO), and free iron. Subsequently, biliverdin is converted tobilirubin via biliverdin reductase, and the free iron is sequesteredinto ferritin (the production of which is induced by the free iron).

CO is recognized as an important signaling molecule (Verma et al.,Science 259:381-384, 1993). It has been suggested that carbon monoxideacts as a neuronal messenger molecule in the brain (Verma et al.) and asa neuro-endocrine modulator in the hypothalamus (Pozzoli et al.,Endocrinology 735:2314-2317, 1994). Like nitric oxide, CO is a smoothmuscle relaxant (Utz et al., Biochem Pharmacol. 47:195-201, 1991;Christodoulides et al., Circulation 97:2306-9, 1995) and inhibitsplatelet aggregation (Mansouri et al., Thromb Haemost. 48:286-8, 1982).Inhalation of low levels of CO has been shown to have anti-inflammatoryeffects in some models.

Efficient DNA damage repair and checkpoint mechanisms are criticalcomponents of normal cellular function to maintain the integrity ofgenomic DNA (Garinis et al., Nat Cell Biol 10 (11), 1241 (2008); Lombardet al., Cell 120 (4), 497 (2005)). DNA lesions are induced in responseto UV or ionizing radiation as well as many chemicals includingendogenous metabolites and reactive oxygen species (ROS). DNA repairpathways include repair of damaged bases or single-strand DNA breaks(base excision repair) and repair of double strand DNA breaks (DSB)including homologous recombination (HR) or non-homologous end-joining(NHEJ).

SUMMARY

The present invention is based, in part, on the discovery thatadministration of CO can protect against the development of DNA damagein cells (and/or enhance DNA repair) that may result from exposure toDNA-damaging levels of radiation. Methods described herein are useful,e.g., for treating subjects who have been exposed, are suspected to havebeen exposed, or will be exposed, to radiation, e.g., from a nuclearreactor or a nuclear weapon, from a radiation therapy (e.g., externalbeam radiation therapy (e.g., x-rays and/or gamma rays) or radioactivepharmaceutical compound), and/or from radioactive materials.

Accordingly, the present disclosure features methods of administering agenotoxic treatment, e.g., administration of a genotoxicchemotherapeutic agent, radiotherapy, and hyperthermia therapy, to apatient. The method includes administering the genotoxic treatment tothe patient, and before, during, and/or after administering thegenotoxic treatment, administering to the patient a pharmaceuticalcomposition comprising carbon monoxide in an amount effective to protectcells of the patient and/or reduce DNA damage in the patient.

In one aspect, methods of reducing radiation-induced DNA damage in asubject are provided. The methods include identifying a subject who willbe, is being, or has been exposed to DNA-damaging levels of radiation;and administering to the subject a pharmaceutical composition comprisingan amount of carbon monoxide effective to reduce radiation-induced DNAdamage in the subject.

In one embodiment, the subject will be, is being, or has been exposed toDNA-damaging levels of radiation in their occupation, where theoccupation is selected from the group consisting of a health careworker, miner, nuclear energy worker, and airline crew member. In oneembodiment, the subject will be, is being, or has been exposed toDNA-damaging levels of radiation from a nuclear reactor or nuclearweapon.

In one embodiment, the pharmaceutical composition is administeredbefore, while, and/or after the subject has been exposed to DNA-damaginglevels of radiation.

In one embodiment, the pharmaceutical composition is in gaseous form andis administered to the patient by inhalation. In one embodiment, thepharmaceutical composition is in liquid form and is administered to thepatient orally. In one embodiment, the pharmaceutical composition isadministered directly to the abdominal cavity of the patient. In oneembodiment, the pharmaceutical composition comprises a carbonmonoxide-releasing compound. In one embodiment, the pharmaceuticalcomposition is administered by an artificial lung. In one embodiment,the pharmaceutical composition is administered by an extracorporealmembrane gas exchange device.

In another aspect, methods of administering a genotoxic treatment to apatient are featured. The methods include (a) administering thegenotoxic treatment to the patient; and (b) before, during, or afterstep (a), administering to the patient a pharmaceutical compositioncomprising carbon monoxide in an amount effective to protect cells ofthe patient, wherein the genotoxic treatment is radiotherapy orhyperthermia therapy.

In one embodiment, the pharmaceutical composition is administeredbefore, during, and/or after step (a). In one embodiment, thepharmaceutical composition is in gaseous form and is administered to thepatient by inhalation. In one embodiment, the pharmaceutical compositionis in liquid form and is administered to the patient orally. In oneembodiment, the pharmaceutical composition is administered directly tothe abdominal cavity of the patient. In one embodiment, thepharmaceutical composition comprises a carbon monoxide-releasingcompound. In one embodiment, the pharmaceutical composition isadministered by an artificial lung. In one embodiment, thepharmaceutical composition is administered by an extracorporeal membranegas exchange device.

In yet another aspect, methods of ameliorating age-related damage to DNAin a patient are provided. The methods include administering to thepatient a pharmaceutical composition comprising an amount of carbonmonoxide effective to ameliorate age-related damage to DNA in thepatient.

In one embodiment, the the pharmaceutical composition is in gaseous formand is administered to the patient by inhalation. In one embodiment, thepharmaceutical composition is in liquid form and is administered to thepatient orally. In one embodiment, the pharmaceutical composition isadministered directly to the abdominal cavity of the patient. In oneembodiment, the pharmaceutical composition comprises a carbonmonoxide-releasing compound. In one embodiment, the pharmaceuticalcomposition is administered by an artificial lung. In one embodiment,the pharmaceutical composition is administered by an extracorporealmembrane gas exchange device.

The subject or patient can be an animal, human or non-human. Forexample, the patient can be any mammal, e.g., humans, other primates,pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters,cows, horses, cats, dogs, sheep and goats. The DNA damage can be theresult of, or a person may be considered at risk for DNA damage becauseof, any of a number of factors. The pharmaceutical composition can be inany form, e.g., gaseous or liquid form.

The pharmaceutical composition can be administered to the patient by anymethod known in the art for administering gases and/or liquids topatients, e.g., via inhalation, insufflation, infusion, injection,and/or ingestion. In one embodiment of the present invention, thepharmaceutical composition is administered to the patient by inhalation.In another embodiment, the pharmaceutical composition is administered tothe patient orally. In still another embodiment, the pharmaceuticalcomposition is administered directly to the abdominal cavity of thepatient. In yet another embodiment, the pharmaceutical composition isadministered by an artificial lung or an extracorporeal membrane gasexchange device.

The present disclosure also features methods of treating, preventing, orreducing the risk of, DNA damage in a patient. The methods includeidentifying a patient diagnosed as suffering from or at risk for DNAdamage (e.g., a patient diagnosed as suffering from or at risk for DNAdamage), and administering to the patient a pharmaceutical compositioncomprising an amount of carbon monoxide effective to treat DNA damage inthe patient.

The present disclosure also features methods of inhibiting or reducingaging or cellular senescence in a patient. The method includeidentifying a patient in need of inhibition or reduction of aging orcellular senescence and administering to the patient a pharmaceuticalcomposition comprising an effective amount of carbon monoxide.

In another embodiment, the method further includes administering to thepatient at least one of the following treatments: inducing HO-1 orferritin in the patient; expressing recombinant HO-1 or ferritin in thepatient; and administering a pharmaceutical composition comprising HO-1,bilirubin, biliverdin, ferritin, or apoferritin, iron, desferoxamine, oriron dextran to the patient. Also contemplated is use of CO and any ofthe above-listed agents in the preparation of a medicament for treatmentor prevention of DNA damage.

In another aspect, the invention features a method of treating orpreventing DNA damage in a patient, which includes identifying a patientsuffering from or at risk for DNA damage (e.g., a patient diagnosed assuffering from or at risk for DNA damage), providing a vessel containinga pressurized gas comprising carbon monoxide gas, releasing thepressurized gas from the vessel to form an atmosphere comprising carbonmonoxide gas, and exposing the patient to the atmosphere, wherein theamount of carbon monoxide in the atmosphere is sufficient to treat DNAdamage in the patient.

Also contemplated is use of CO in the preparation of a medicament, e.g.,a gaseous or liquid medicament, for use in the treatment or preventionof DNA damage.

Also within the invention is the use of CO in the manufacture of amedicament for treatment or prevention of DNA damage. The medicament canbe used in a method for treating DNA damage in a patient suffering fromor at risk for DNA damage in accordance with the methods describedherein. The medicament can be in any form described herein, e.g., aliquid, solid (CO-releasing compound tablet, enema, etc.), or gaseous COcomposition.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Suitable methods and materialsare described below, although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C are a panel of three figures showing the levels of H2AXγ intissues from hmox1^(−/−) mice. A-B. An immunohistochemical analysis ofH2AXγ in kidney, lung, liver, and spleen of wild type (Hmox1^(+/+)) andHmox1^(−/−) mice. C. An immunoblot with antibodies against H2AXγ andHO-1 in the spleen lysates of wild type (+/+), Hmox1^(−/+) (+/−) andHmox1^(−/−) (−/−) mice. Representative Western blot is shown out of twoexperiments.

FIGS. 2A-C are a series of three figures depicting a lack of HO-1results in altered H2AXγ activation in response to DNA damage. A. Apanel of photomicrographs showing fibroblasts from Hmox1^(+/+) andHmox1^(−/−) l mice isolated and treated with camptothecin (1 μg/ml) ordoxorubicin (10 μg/ml) for 1 hour and stained with antibodies againstH2AXγ. Scale bar: 25 μm. B-C. An immunoblot analysis of H2AXγ in thelysates of Hmox1^(+/+), Hmox1^(−/+) and Hmox1^(−/−) fibroblasts thatwere treated with doxorubicin. Representative figure is shown in B;quantitation is presented in C. Data are representative for at least 2independent experiments. *p<0.05; 4 hours versus control in Hmox1^(+/+)fibroblasts. #p<0.05; Hmox1^(−/−) versus Hmox1^(+/+).

FIGS. 3A-D are a panel of immunoblots showing DNA repair signaling inthe absence of HO-1. A-B. HEK293 cells with stable knockdown of HO-1(mirHO-1) and control cells treated with camptothecin. C. An immunoblotanalysis in HEK mirHO-1 and control cells treated with doxorubicin. Dataare representative for two experiments. D. An immunoblot analysis withantibodies against P-ATM, P-Brca1, P-H2AX, P-p53 in the lysates ofHEK293 mirHO-1 and control cells treated with 10 Gy g-irradiation andharvested 5′-1 h after irradiation. Data are representative for twoindependent experiments in triplicates.

FIGS. 4A-G are a panel of figures showing HO-1/CO induces HR-mediatedDNA repair. A. A photomicrograph showing HO-1 levels as measured byWestern blot. B-D. A photomicrograph and graphs showing the number ofGFP-positive U20S-SCR reporter cells as measured by fluorescencemicroscope (B) and flow cytometry (C-D). Data are representative for atleast five experiments in duplicate. Data±SD are shown. p<0.05*; COversus air. Scale bar: 25 μm. E-G. A photomicrograph and graphs showingthe level of GFP-positive cells measured by fluorescence microscopy (E)or flow cytometry (F-G). Representative data are shown and arerepresentative for three independent experiments; averages±SD. Scalebar: 25 μm.

FIGS. 5A-F are a series of figures showing CO induces activation of DNArepair signaling. A-B. Immunoblots with antibody against P-Brca1, P-ATM(A) and P-ATR (B) in the lysates of PC3 and HEK cells that were treatedwith CO. Data are representative for 3 independent experiments. C-D.Graphs showing the levels of GFP-positive cells were measured by flowcytometry. *p<0.05 CO versus Air; #p<0.05 CO+CGK733 versus CO. E. A bargraph showing the levels of GFP-positive cells were measured by flowcytometry. *p<0.05, CO versus Air, **p<0.01 CO+KU55933 versus CO; (−)untransfected cells. F. A bar graph showing the level of GFP-positivecells was measured by flow cytometry. Data are representative for 3independent experiments in duplicates. *p<0.05 HO-1 versus C, #p<0.05HO-1+CGK733 versus HO-1.

FIGS. 6A-F are a panel of photomicrographs and bar graphs showing COblocks DNA damage in tissues of mice treated with doxorubicin. A-E. Aseries of 15 photomicrographs of immunoblots with antibody against H2AXγin colon (A), kidney (B), lung (C), liver (D), and spleen (E).Magnification 20× for colon and kidney; magnification 40× for spleen,liver, lung. #p<0.05, doxorubicin versus control; *p<0.05 CO+doxorubicinversus Air+doxorubicin; **p<0.001 CO+doxorubicin versus Air+doxorubicin.F. A bar graph depicting quantitation of H2AXγ staining in the tissues.Control (Air or CO), n=3, Doxorubicin±CO, n=5. Averages±SD of number ofH2AXγ positive cells per FOV are presented.

FIGS. 7A-G are a series of photomicrographs and graphs showing COdecreases the lethal dose irradiation induced damage in the tissues.A-B. An immunoblot (A) and immunohistochemistry (B) with antibodyagainst HO-1 in the tissues from mice irradiated with 10 Gy. Liver andspleens after 1 and 3 days post-irradiation are shown in B. C-D. Animmunohistochemical analysis of P-H2AX in the spleens and intestines ofmice pretreated with Air or CO 1 hour prior lethal dose of irradiationand treated daily with CO for 11 days. Representative pictures are shownin C and quantitation of the number of H2AXg positive cells in D. n=3-4views/sections; n=3-4 mice/group. E-F. An immunofluorescence staining ofP-ATM and P-p53 in mononuclear blood cells of mice that were lethallyirradiated and treated with CO/Air for 1 hour prior to irradiation.Representative pictures and quantitation of number of positive cells perfield of view is shown. n=3-4/group. G. A graphs showing survival ofmice after marginal bone marrow transplantation from H2ax^(−/−) andH2ax^(+/+) mice to the wild type recipients. n=5-10/per group.

FIG. 8 is a bar graph depicting beta-galactosidase activity in zebrafish embryos as an accepted measure of senesence.

FIGS. 9A-B are a series of two bar graphs showing CO exposure increasesgene expression of SIRT1 (9A) and TERT (9B) in vivo.

FIG. 10 is a bar graph showing change in expression of telomerase inlung, and topoisomerase and p16 in intestine. Results represent mean±SDof at least 3 mice/group, *p<0.05.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery that COadministration ameliorates DNA damage.

The term “carbon monoxide” (or “CO”) as used herein describes molecularcarbon monoxide in its gaseous state, compressed into liquid form, ordissolved in aqueous solution. The terms “carbon monoxide composition”and “pharmaceutical composition comprising carbon monoxide” is usedthroughout the specification to describe a gaseous, solid, or liquidcomposition containing carbon monoxide that can be administered to apatient and/or an organ by routes including orally, intravenously,subcutaneously, intramuscularly, and topically. The skilled practitionerwill recognize which form of the pharmaceutical composition, e.g.,gaseous, liquid, or both gaseous and liquid forms, is preferred for agiven application.

The terms “effective amount” and “effective to treat,” as used herein,refer to an amount or concentration of carbon monoxide utilized forperiod of time (including acute or chronic administration and periodicor continuous administration) that is effective within the context ofits administration for causing an intended effect or physiologicaloutcome. Effective amounts of carbon monoxide for use in the presentinvention include, for example, amounts that prevent DNA damage, reducethe risk of DNA damage, reduce the symptoms of DNA damage, improve theoutcome of genotoxic treatments, extend life, increase life span, and/orprevent aging.

For gases, effective amounts of carbon monoxide generally fall withinthe range of about 0.0000001% to about 0.3% by weight, e.g., 0.0001% toabout 0.25% by weight, preferably at least about 0.001%, e.g., at least0.005%, 0.010%, 0.02%, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.10%,0.15%, 0.20%, 0.22%, or 0.24% by weight carbon monoxide. Preferredranges include, e.g., 0.001% to about 0.24%, about 0.005% to about0.22%, about 0.005% to about 0.05%, about 0.010% to about 0.20%, about0.02% to about 0.15%, about 0.025% to about 0.10%, or about 0.03% toabout 0.08%, or about 0.04% to about 0.06%. For liquid solutions of CO,effective amounts generally fall within the range of about 0.0001 toabout 0.0044 g CO/100 g liquid, e.g., at least 0.0001, 0.0002, 0.0004,0.0006, 0.0008, 0.0010, 0.0013, 0.0014, 0.0015, 0.0016, 0.0018, 0.0020,0.0021, 0.0022, 0.0024, 0.0026, 0.0028, 0.0030, 0.0032, 0.0035, 0.0037,0.0040, or 0.0042 g CO/100 g aqueous solution. Preferred ranges include,e.g., about 0.0010 to about 0.0030 g CO/100 g liquid, about 0.0015 toabout 0.0026 g CO/100 g liquid, or about 0.0018 to about 0.0024 g CO/100g liquid. A skilled practitioner will appreciate that amounts outside ofthese ranges may be used, depending upon the application. The skilledpractitioner will also appreciate that amounts of CO derived from aCO-RM or CO-Hb that would achieve the same doses in the body when givenas a gas can be administered.

The term and “subject” and “patient” are used interchangeably throughoutthe specification to describe an animal, human or non-human, to whomtreatment according to the methods of the present invention is provided.Veterinary applications are contemplated by the present invention. Theterm includes but is not limited to mammals, e.g., humans, otherprimates, pigs, rodents such as mice and rats, rabbits, guinea pigs,hamsters, cows, horses, cats, dogs, sheep and goats. The term“treat(ment),” is used herein to describe delaying the onset of,inhibiting, or alleviating the effects of a condition, e.g., DNA damage,in a patient.

The term “DNA damage” is an art-recognized term and is used herein torefer to chemical changes to DNA, e.g., damaged (oxidized, alkylated,hydrolyzed, adducted, or cross-linked) bases, single-stranded DNAbreaks, and double-stranded DNA breaks.

Causative agents of DNA damage include, for example, ultraviolet light,ionizing radiation (X-rays, gamma rays, alpha particles), aging andaging-related disorders (e.g., Hutchinson-Gilford Progeria Syndrome,Werner's syndrome, Cockayne's syndrome, or xeroderma pigmentosum), andgenotoxic or mutagenic agents, e.g., reactive oxygen species, baseanalogs, deaminating agents (e.g., nitrous acid), intercalating agents(e.g., ethidium bromide), alkylating agents (e.g., ethylnitrosourea),alkaloids (e.g., Vinca alkaloids), bromines, sodium azide, psoralen, andbenzene. Exemplary genotoxic agents used in cancer therapy includebusulfan, bendamustine, carboplatin, carmustine, chlorambucil,cisplatin, cyclophosphamide, dacarbazine, daunorubicin, decitabine,doxorubicin, epirubicin, etoposide, idarubicin, ifosfamide, irinotecan,lomustine, mechlorethamine, melphalan, mitomycin C, mitoxantrone,oxaliplatin, temozolomide, and topotecan.

In some cases, a subject or patient suffering from or at risk for DNAdamage has been exposed to or is likely to be exposed to ionizingradiation, e.g., at an acute dose of at least or about 0.1 Gy, e.g., atleast or about 0.2 Gy, at least or about 0.5 Gy, at least or about 1 Gy,at least or about 2 Gy, at least or about 4 Gy, at least or about 5 Gy,at least or about 6 Gy, at least or about 7 Gy, at least or about 8 Gy,at least or about 10 Gy, at least or about 20 Gy, at least or about 30Gy, at least or about 40 Gy, at least or about 50 Gy, or at least orabout 100 Gy. Skilled practitioners will appreciate what levels ofradiation damage DNA. In some cases, a subject will be, is being, or hasbeen exposed to DNA-damaging levels of radiation, e.g., at least orabout 0.1 Gy, e.g., at least or about 0.2 Gy, at least or about 0.5 Gy,at least or about 1 Gy, at least or about 2 Gy, at least or about 4 Gy,at least or about 5 Gy, at least or about 6 Gy, at least or about 7 Gy,at least or about 8 Gy, at least or about 10 Gy, at least or about 20Gy, at least or about 30 Gy, at least or about 40 Gy, at least or about50 Gy, or at least or about 100 Gy. The subject can be exposed toDNA-damaging levels of radiation through their occupation, e.g., ahealth care worker, miner, nuclear energy worker, and airline crewmember, from a nuclear reactor, or from nuclear weapon, e.g., duringwarfare and/or an act of terrorism. The pharmaceutical composition canbe administered before, while, and/or after the subject has been exposedto DNA-damaging levels of radiation.

In some cases, a patient suffering from or at risk for DNA damage willbe suffering from a burn. A patient suffering from or at risk for DNAdamage can be one undergoing one or more genotoxic treatments, e.g.,chemotherapy with a genotoxic agent, radiotherapy, or hyperthermiatherapy. A patient suffering from or at risk for DNA damage may have adeleterious genetic defect or mutation. In some cases, the presentmethods ameliorate age-related damage to DNA in a patient.

In some cases, aging is a consequence of unrepaired DNA damageaccumulation. Age-related damage to DNA results in DNA alteration thathas an abnormal structure. Although both mitochondrial and nuclear DNAdamage can contribute to aging, nuclear DNA is the main subject of thisanalysis. Nuclear DNA damage can contribute to aging either indirectly(by increasing apoptosis or cellular senescence) or directly (byincreasing cell dysfunction).

In humans, DNA damage occurs frequently, and DNA repair processes haveevolved to compensate. On average, approximately 800 DNA lesions occurper hour in each cell, or about 19,200 per cell per day. In any cell,some DNA damage may remain despite the action of repair processes.Accumulation of unrepaired DNA damage is more prevalent in certain typesof cells, particularly in non-replicating or slowly replicating cells,which cannot rely on DNA repair mechanisms associated with DNAreplication such as those in the brain, skeletal, and cardiac muscle.Older patients, e.g., at least or about 30 years old, at least or about35 years old, at least or about 40 years old, at least or about 45 yearsold, at least or about 50 years old, at least or about 55 years old, atleast or about 60 years old, at least or about 65 years old, at least orabout 70 years old, at least or about 75 years old, or at least or about80 years old, typically accumulate more age-related damage to DNA andcan benefit from treatment to ameliorate age-related damage to DNA.

Skilled practitioners will appreciate that a patient can be diagnosed bya physician as suffering from or at risk for DNA damage by any methodknown in the art. Individuals considered at risk for developing DNAdamage may benefit particularly from the invention, primarily becauseprophylactic treatment can begin before there is any evidence of DNAdamage. Individuals “at risk” include, e.g., subjects exposed toenvironmental, occupational, or therapeutic genotoxic agents. Theskilled practitioner will appreciate that a patient can be determined tobe at risk for DNA damage by a physician's diagnosis.

Amounts of CO effective to treat DNA damage can be administered to apatient on the day the patient is diagnosed as suffering from DNA damageor any condition associated with DNA damage, or as having any riskfactor associated with an increased likelihood that the patient willdevelop DNA damage (e.g., that the patient has recently been, is being,or will be exposed to a genotoxic agent). Patients can inhale CO atconcentrations ranging from 10 ppm to 1000 ppm, e.g., about 100 ppm toabout 800 ppm, about 150 ppm to about 600 ppm, or about 200 ppm to about500 ppm. Preferred concentrations include, e.g., about 30 ppm, 50 ppm,75 ppm, 100 ppm, 125 ppm, 200 ppm, 250 ppm, 500 ppm, 750 ppm, or about1000 ppm. CO can be administered to the patient intermittently orcontinuously. CO can be administered for about 1, 2, 4, 6, 8, 10, 12,14, 18, or 20 days, or greater than 20 days, e.g., 1 2, 3, 5, or 6months, or until the patient no longer exhibits symptoms of DNA damage,or until the patient is diagnosed as no longer being at risk for DNAdamage. In a given day, CO can be administered continuously for theentire day, or intermittently, e.g., a single whiff of CO per day (wherea high concentration is used), or for up to 23 hours per day, e.g., upto 20, 15, 12, 10, 6, 3, or 2 hours per day, or up to 1 hour per day. Adosage of CO administered can be converted mathematically to a mg/kgdosing based on time and concentration of administration and a patient'sbody weight.

If the patient needs to be treated with a genotoxic drug (e.g., becauseprescribed by a physician), the patient can be treated with CO (e.g., agaseous CO composition) before, during, and/or after administration ofthe drug. For example, CO can be administered to the patient,intermittently or continuously, starting 0 to 20 days before the drug isadministered (and where multiple doses are given, before each individualdose), e.g., starting at least about 30 minutes, e.g., about 1, 2, 3, 5,7, or 10 hours, or about 1, 2, 4, 6, 8, 10, 12, 14, 18, or 20 days, orgreater than 20 days, before the administration. Alternatively or inaddition, CO can be administered to the patient concurrent withadministration of the drug. Alternatively or in addition, CO can beadministered to the patient after administration of the drug, e.g.,starting immediately after administration, and continuing intermittentlyor continuously for about 1, 2, 3, 5, 7, or 10 hours, or about 1, 2, 5,8, 10, 20, 30, 50, or 60 days, indefinitely, or until a physiciandetermines that administration of CO is no longer necessary (e.g., afterthe genotoxic agent is eliminated from the body or can no longer causeDNA damage).

Administration of CO is further described in U.S. Pat. No. 7,238,469;U.S. Pat. No. 7,678,390; U.S. Pat. No. 7,687,079; U.S. Pat. No.7,981,448; U.S. Pat. No. 7,364,757; US 2004/0258772; US 2004/0052866; US2004/0228930; and US 2004/0131703, each of which is incorporated byreference herein in its entirety.

Preparation of Gaseous Compositions

A carbon monoxide composition may be a gaseous carbon monoxidecomposition. Compressed or pressurized gas useful in the methods of theinvention can be obtained from any commercial source and in any type ofvessel appropriate for storing compressed gas. For example, compressedor pressurized gases can be obtained from any source that suppliescompressed gases, such as oxygen, for medical use. The term “medicalgrade” gas, as used herein, refers to gas suitable for administration topatients as defined herein. The pressurized gas including CO used in themethods of the present invention can be provided such that all gases ofthe desired final composition (e.g., CO, He, NO, CO₂, O₂, N₂) are in thesame vessel, except that NO and O₂ cannot be stored together.Optionally, the methods of the present invention can be performed usingmultiple vessels containing individual gases. For example, a singlevessel can be provided that contains carbon monoxide, with or withoutother gases, the contents of which can be optionally mixed with room airor with the contents of other vessels, e.g., vessels containing oxygen,nitrogen, carbon dioxide, compressed air, or any other suitable gas ormixtures thereof.

Gaseous compositions administered to a patient according to the presentinvention typically contain 0% to about 79% by weight nitrogen, about21% to about 100% by weight oxygen and about 0.0000001% to about 0.3% byweight (corresponding to about 1 ppb or 0.001 ppm to about 3,000 ppm)carbon monoxide. Preferably, the amount of nitrogen in the gaseouscomposition is about 79% by weight, the amount of oxygen is about 21% byweight and the amount of carbon monoxide is about 0.0001% to about 0.25%by weight, preferably at least about 0.001%, e.g., at least about0.005%, 0.010%, 0.02%, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.10%,0.15%, 0.20%, 0.22%, or 0.24% by weight. Preferred ranges of carbonmonoxide include about 0.005% to about 0.24%, about 0.01% to about0.22%, about 0.015% to about 0.20%, about 0.08% to about 0.20%, andabout 0.025% to about 0.1% by weight. It is noted that gaseous carbonmonoxide compositions having concentrations of carbon monoxide greaterthan 0.3% (such as 1% or greater) may be used for short periods (e.g.,one or a few breaths), depending upon the application.

A gaseous carbon monoxide composition may be used to create anatmosphere that comprises carbon monoxide gas. An atmosphere thatincludes appropriate levels of carbon monoxide gas can be created, forexample, by providing a vessel containing a pressurized gas comprisingcarbon monoxide gas, and releasing the pressurized gas from the vesselinto a chamber or space to form an atmosphere that includes the carbonmonoxide gas inside the chamber or space. Alternatively, the gases canbe released into an apparatus that culminates in a breathing mask orbreathing tube, thereby creating an atmosphere comprising carbonmonoxide gas in the breathing mask or breathing tube, ensuring thepatient is the only person in the room exposed to significant levels ofcarbon monoxide.

Carbon monoxide levels in an atmosphere can be measured or monitoredusing any method known in the art. Such methods include electrochemicaldetection, gas chromatography, radioisotope counting, infraredabsorption, colorimetry, and electrochemical methods based on selectivemembranes (see, e.g., Sunderman et al., Clin. Chem. 28:2026-2032, 1982;Ingi et al., Neuron 16:835-842, 1996). Sub-parts per million carbonmonoxide levels can be detected by, e.g., gas chromatography andradioisotope counting. Further, it is known in the art that carbonmonoxide levels in the sub-ppm range can be measured in biologicaltissue by a midinfrared gas sensor (see, e.g., Morimoto et al., Am. J.Physiol. Heart. Circ. Physiol 280:H482-H488, 2001). Carbon monoxidesensors and gas detection devices are widely available from manycommercial sources.

Preparation of Liquid Compositions

A carbon monoxide composition may also be a liquid carbon monoxidecomposition. A liquid can be made into a carbon monoxide composition byany method known in the art for causing gases to become dissolved inliquids. For example, the liquid can be placed in a so-called “CO₂incubator” and exposed to a continuous flow of carbon monoxide,preferably balanced with carbon dioxide, until a desired concentrationof carbon monoxide is reached in the liquid. As another example, carbonmonoxide gas can be “bubbled” directly into the liquid until the desiredconcentration of carbon monoxide in the liquid is reached. The amount ofcarbon monoxide that can be dissolved in a given aqueous solutionincreases with decreasing temperature. As still another example, anappropriate liquid may be passed through tubing that allows gasdiffusion, where the tubing runs through an atmosphere comprising carbonmonoxide (e.g., utilizing a device such as an extracorporeal membraneoxygenator). The carbon monoxide diffuses into the liquid to create aliquid carbon monoxide composition.

It is likely that such a liquid composition intended to be introducedinto a living animal will be at or about 37° C. at the time it isintroduced into the animal.

The liquid can be any liquid known to those of skill in the art to besuitable for administration to patients (see, for example, OxfordTextbook of Surgery, Morris and Malt, Eds., Oxford University Press(1994)). In general, the liquid will be an aqueous solution. Examples ofsolutions include Phosphate Buffered Saline (PBS), Celsior™, Perfadex™,Collins solution, citrate solution, and University of Wisconsin (UW)solution (Oxford Textbook of Surgery, Morris and Malt, Eds., OxfordUniversity Press (1994)). In one embodiment of the present invention,the liquid is Ringer's Solution, e.g., lactated Ringer's Solution, orany other liquid that can be used infused into a patient. In anotherembodiment, the liquid includes blood, e.g., whole blood.

Any suitable liquid can be saturated to a set concentration of carbonmonoxide via gas diffusers. Alternatively, pre-made solutions that havebeen quality controlled to contain set levels of carbon monoxide can beused. Accurate control of dose can be achieved via measurements with agas permeable, liquid impermeable membrane connected to a carbonmonoxide analyzer. Solutions can be saturated to desired effectiveconcentrations and maintained at these levels.

Treatment of Patients with Carbon Monoxide Compositions

A patient can be treated with a carbon monoxide composition by anymethod known in the art of administering gases and/or liquids topatients. Carbon monoxide compositions can be administered to a patientdiagnosed with, or determined to be at risk for, DNA damage. The presentinvention contemplates the systemic administration of liquid or gaseouscarbon monoxide compositions to patients (e.g., by inhalation and/oringestion), and the topical administration of the compositions to thepatient (e.g., by introduction into the abdominal cavity).

Systemic Delivery of Carbon Monoxide

Gaseous carbon monoxide compositions can be delivered systemically to apatient, e.g., a patient diagnosed with, or determined to be at risk forDNA damage. Gaseous carbon monoxide compositions are typicallyadministered by inhalation through the mouth or nasal passages to thelungs, where the carbon monoxide is readily absorbed into the patient'sbloodstream. The concentration of active compound (CO) utilized in thetherapeutic gaseous composition will depend on absorption, distribution,inactivation, and excretion (generally, through respiration) rates ofthe carbon monoxide as well as other factors known to those of skill inthe art. It is to be further understood that for any particular subject,specific dosage regimens should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the concentration ranges set forth herein are exemplary only andare not intended to limit the scope or practice of the claimedcomposition. Treatments can be monitored and CO dosages can be adjustedto ensure optimal treatment of the patient. Acute, sub-acute and chronicadministration of carbon monoxide is contemplated by the presentinvention, depending upon, e.g., the severity or persistence of DNAdamage in the patient. Carbon monoxide can be delivered to the patientfor a time (including indefinitely) sufficient to treat the conditionand exert the intended pharmacological or biological effect.

The following are examples of some methods and devices that can beutilized to administer gaseous carbon monoxide compositions to patients.

Ventilators

Medical grade carbon monoxide (concentrations can vary) can be purchasedmixed with air or another oxygen-containing gas in a standard tank ofcompressed gas (e.g., 21% O₂, 79% N₂). It is non-reactive, and theconcentrations that are required for the methods of the presentinvention are well below the combustible range (10% in air). In ahospital setting, the gas presumably will be delivered to the bedsidewhere it will be mixed with oxygen or house air in a blender to adesired concentration in ppm (parts per million). The patient willinhale the gas mixture through a ventilator, which will be set to a flowrate based on patient comfort and needs. This is determined by pulmonarygraphics (i.e., respiratory rate, tidal volumes etc.). Fail-safemechanism(s) to prevent the patient from unnecessarily receiving greaterthan desired amounts of carbon monoxide can be designed into thedelivery system. The patient's carbon monoxide level can be monitored bystudying (1) carboxyhemoglobin (COHb), which can be measured in venousblood, and (2) exhaled carbon monoxide collected from a side port of theventilator. Carbon monoxide exposure can be adjusted based upon thepatient's health status and on the basis of the markers. If necessary,carbon monoxide can be washed out of the patient by switching to 100% O₂inhalation. Carbon monoxide is not metabolized; thus, whatever isinhaled will ultimately be exhaled except for a very small percentagethat is converted to CO₂. Carbon monoxide can also be mixed with anylevel of O₂ to provide therapeutic delivery of carbon monoxide withoutconsequential hypoxic conditions.

Face Mask and Tent

A carbon monoxide-containing gas mixture is prepared as above to allowpassive inhalation by the patient using a facemask or tent. Theconcentration inhaled can be changed and can be washed out by simplyswitching over to 100% O₂. Monitoring of carbon monoxide levels wouldoccur at or near the mask or tent with a fail-safe mechanism that wouldprevent too high of a concentration of carbon monoxide from beinginhaled.

Portable Inhaler

Compressed carbon monoxide can be packaged into a portable inhalerdevice and inhaled in a metered dose, for example, to permitintermittent treatment of a recipient who is not in a hospital setting.Different concentrations of carbon monoxide could be packaged in thecontainers. The device could be as simple as a small tank (e.g., under 5kg) of appropriately diluted CO with an on-off valve and a tube fromwhich the patient takes a whiff of CO according to a standard regimen oras needed.

Intravenous Artificial Lung

An artificial lung (a catheter device for gas exchange in the blood)designed for O₂ delivery and CO₂ removal can be used for carbon monoxidedelivery. The catheter, when implanted, resides in one of the largeveins and would be able to deliver carbon monoxide at givenconcentrations either for systemic delivery or at a local site. Thedelivery can be a local delivery of a high concentration of carbonmonoxide for a short period of time at the site of the procedure (thishigh concentration would rapidly be diluted out in the bloodstream), ora relatively longer exposure to a lower concentration of carbon monoxide(see, e.g., Hattler et al., Artif. Organs 18 (11):806-812 (1994); andGolob et al., ASAIO J., 47 (5):432-437 (2001)).

Normobaric Chamber

In certain instances, it may be desirable to expose the whole patient tocarbon monoxide. The patient would be inside an airtight chamber thatwould be flooded with carbon monoxide (at a level that does not endangerthe patient, or at a level that poses an acceptable risk without therisk of bystanders being exposed. Upon completion of the exposure, thechamber could be flushed with air (e.g., 21% O₂, 79% N₂) and samplescould be analyzed by carbon monoxide analyzers to ensure no carbonmonoxide remains before allowing the patient to exit the exposuresystem.

Systemic Delivery of Liquid CO Compositions

The present invention further contemplates that aqueous solutionscomprising carbon monoxide can be created for systemic delivery to apatient, e.g., for oral delivery and/or by infusion into the patient,e.g., intravenously, intra-arterially, intraperitoneally, and/orsubcutaneously. For example, liquid CO compositions, such asCO-saturated Ringer's Solution, can be infused into a patient sufferingfrom or at risk for DNA damage. Alternatively or in addition,CO-partially or completely saturated whole (or partial) blood can beinfused into the patient. CO can also be administered as a CO-RM orCO-saturated Hb (artificial, e.g. pegylated or non-pegylated; also couldbe any formulation of Hb that can be saturated with CO and then infusedinto the patient.)

The present invention also contemplates that agents capable ofdelivering doses of gaseous CO compositions or liquid CO compositionscan be utilized (e.g., CO-releasing gums, creams, ointments, lozenges,or patches).

Topical Treatment of Organs with Carbon Monoxide

Alternatively or in addition, carbon monoxide compositions can beapplied directly to an organ, or to any portion thereof, to ameliorateDNA damage. A gaseous composition can be directly applied to an organ ofa patient by any method known in the art for insufflating gases into apatient. For example, gases, e.g., carbon dioxide, are often insufflatedinto the abdominal cavity of patients to facilitate examination duringlaproscopic procedures (see, e.g., Oxford Textbook of Surgery, Morrisand Malt, Eds., Oxford University Press (1994)). The skilledpractitioner will appreciate that similar procedures could be used toadminister carbon monoxide compositions directly to an organ of apatient.

Aqueous carbon monoxide compositions can also be administered topicallyto an organ of a patient to ameliorate DNA damage. Aqueous forms of thecompositions can be administered by any method known in the art foradministering liquids to patients. As with gaseous compositions, aqueouscompositions can be applied directly to the organ. For example, liquids,e.g., saline solutions containing dissolved CO, can be injected into theabdominal cavity of patients during laproscopic procedures. The skilledpractitioner will appreciate that similar procedures could be used toadminister liquid carbon monoxide compositions directly to an organ of apatient. Further, an in situ exposure can be carried out by flushing theorgan or a portion thereof with a liquid carbon monoxide composition(see Oxford Textbook of Surgery, Morris and Malt, Eds., OxfordUniversity Press (1994)).

Use of Heme Oxygenase-1, Other Compounds, and Other Treatments for DNADamage

Also contemplated by the present invention is the induction orexpression of heme oxygenase-1 (HO-1) in conjunction with administrationof CO. For example, HO-1 can be induced in a patient suffering from orat risk for DNA damage. As used herein, the term “induce(d)” means tocause increased production of a protein, e.g., HO-1, in isolated cellsor the cells of a tissue, organ or animal using the cells' ownendogenous (e.g., non-recombinant) gene that encodes the protein.

HO-1 can be induced in a patient by any method known in the art. Forexample, production of HO-1 can be induced by hemin/heme arginate, byiron protoporphyrin, or by cobalt protoporphyrin. A variety of non-hemeagents including heavy metals, cytokines, hormones, NO, COCl₂, endotoxinand heat shock are also strong inducers of HO-1 expression (Choi et al.,Am. J. Respir. Cell Mol. Biol. 15:9-19, 1996; Maines, Annu. Rev.Pharmacol. Toxicol. 37:517-554, 1997; and Tenhunen et al., J. Lab. Clin.Med. 75:410-421, 1970). HO-1 is also highly induced by a variety ofagents causing oxidative stress, including hydrogen peroxide,glutathione depletors, UV irradiation, endotoxin and hyperoxia (Choi etal., Am. J. Respir. Cell Mol. Biol. 15:9-19, 1996; Maines, Annu. Rev.Pharmacol. Toxicol. 37:517-554, 1997; and Keyse et al., Proc. Natl.Acad. Sci. USA 86:99-103, 1989). A “pharmaceutical compositioncomprising an inducer of HO-1” means a pharmaceutical compositioncontaining any agent capable of inducing HO-1 in a patient, e.g., any ofthe agents described above, e.g., NO, hemin, iron protoporphyrin, and/orcobalt protoporphyrin.

HO-1 expression in a cell can be increased via gene transfer. As usedherein, the term “express(ed)” means to cause increased production of aprotein, e.g., HO-1 or ferritin, in isolated cells or the cells of atissue, organ or animal using an exogenously administered gene (e.g., arecombinant gene). The HO-1 or ferritin is preferably of the samespecies (e.g., human, mouse, rat, etc.) as the recipient, in order tominimize any immune reaction. Expression could be driven by aconstitutive promoter (e.g., cytomegalovirus promoters) or atissue-specific promoter (e.g., milk whey promoter for mammary cells oralbumin promoter for liver cells). An appropriate gene therapy vector(e.g., retrovirus, adenovirus, adeno-associated virus (AAV), pox (e.g.,vaccinia) virus, human immunodeficiency virus (HIV), the minute virus ofmice, hepatitis B virus, influenza virus, Herpes Simplex Virus-1, andlentivirus) encoding HO-1 or ferritin would be administered to a patientsuffering from or at risk for DNA damage, by mouth, by inhalation, or byinjection. Similarly, plasmid vectors encoding HO-1 or apoferritin canbe administered, e.g., as naked DNA, in liposomes, or in microparticles.

Further, exogenous HO-1 protein can be directly administered to apatient by any method known in the art. Exogenous HO-1 can be directlyadministered in addition, or as an alternative, to the induction orexpression of HO-1 in the patient as described above. The HO-1 proteincan be delivered to a patient, for example, in liposomes, and/or as afusion protein, e.g., as a TAT-fusion protein (see, e.g., Becker-Hapaket al., Methods 24:247-256, 2001).

Alternatively or in addition, any of the products of metabolism by HO-1,e.g., bilirubin, biliverdin, iron, and/or ferritin, can be administeredto a patient in conjunction with CO in order to prevent or treat DNAdamage. Further, the present invention contemplates that iron-bindingmolecules other than ferritin, e.g., desferoxamine (DFO), iron dextran,and/or apoferritin, can be administered to the patient. Further still,the present invention contemplates that enzymes (e.g., biliverdinreductase) that catalyze the breakdown any of these products can beinhibited to create/enhance the desired effect. Any of the above can beadministered, e.g., orally, intravenously, intraperitoneally, or bydirect administration.

The present invention contemplates that compounds that release CO intothe body after administration of the compound (e.g., CO-releasingcompounds, e.g., photoactivatable CO-releasing compounds), e.g.,dimanganese decacarbonyl, tricarbonyldichlororuthenium (II) dimer, andmethylene chloride (e.g., at a dose of between 400 to 600 mg/kg, e.g.,about 500 mg/kg), can also be used in the methods of the presentinvention, as can carboxyhemoglobin and CO-donating hemoglobinsubstitutes. See, e.g., US 2003/0064114, US 2003/0068387, and US2007/0207217.

The above can be administered to a patient in any way, e.g., by oral,intraperitoneal, intravenous, or intraarterial administration as well astopical (including sublingual and suppository) as well as an aerosol tothe lungs. Any of the above compounds can be administered to the patientlocally and/or systemically, and in any combination.

The present invention further contemplates treating/preventing DNAdamage by administering CO to the patient in combination with any otherknown methods or compounds for treating DNA damage, e.g., cessation orreducing administration of genotoxic agents.

The invention is illustrated in part by the following examples, whichare not to be taken as limiting the invention in any way.

EXAMPLES Example 1 Lack of HO-1 Results in Accumulation of γ-H2AX FociIn Vivo

To evaluate the role of HO-1 in DNA damage and repair signaling,immunohistochemical analyses of γ-H2AX staining, a marker of ongoing andchronic DNA damage, was performed on various tissues harvested fromHmox1^(−/−) mice. A low degree of γ-H2AX was observed in the spleens andlungs of wild type animals and nearly undetectable expression in thekidney and liver (FIGS. 1A-B). In contrast, there was a statisticallysignificant greater amount of γ-H2AX foci in Hmox1^(−/−) animalscompared to wild type controls (FIGS. 1A-C). Quantification of thenumber of H2AXγ foci per field of view (20× magnification; n=3-4, fieldsn=5-10 from 3-4 mice) is shown in A. Representative pictures are shownin B. *p=0.03, **p<0.001. Scale bar: 100 μm. These data suggested thateither Hmox1^(−/−) cells are unable to repair broken DNA efficiently orthe extensive oxidative stress in the tissues of Hmox1^(−/−) miceresults in accelerated DNA damage.

To elucidate whether absence of HO-1 results in accelerated accumulationof DNA damage, cells were treated with doxorubicin or camptothecin,which induce DSB or SSB, respectively, as measured by H2AXphosphorylation (FIG. 2). Treatment of Hmox^(+/+) fibroblasts withdoxorubicin or camptothecin resulted in a strong increase inphosphorylation of histone H2AX and formation of multiple γ-H2AX foci(FIGS. 2A-B). Fibroblasts from Hmox1^(−/−) mice also showed similar fociin response to camptothecin as in wild type (FIG. 2A), however incontrast to camptothecin, Hmox1^(−/−) fibroblasts treated withdoxorubicin showed no γ-H2AX foci formation or phosphorylation of γ-H2AXat any time point tested which was otherwise present in wild type cells(FIGS. 2A-C). Representative pictures are shown from at least twoindependent experiments in duplicates. Fibroblasts were treated withdoxorubicin (10 μg/ml) for 2-4 hr. These data suggested that HO-1 canmediate DNA repair responses specifically to DSB that are induced bydoxorubicin and unlike camptothecin which targets DNA topoisomersase I.

Example 2 Absence of HO-1 Results in Decreased DNA Repair Signaling

The direct effects of HO-1 was tested on signaling pathways in responseto the DNA damaging agents, doxorubicin, camptothecin, or irradiation inloss and gain of function studies with HO-1. HEK293 cells express highbasal levels of HO-1 due to presence of large T antigen. HEK293 cellswere transduced with micro-adapted shRNA to stably deplete HO-1 and thenexposed to camptothecin, doxorubicin or irradiation. HEK293 cells withstable knockdown of HO-1 (mirHO-1) and control cells were treated withcamptothecin (1 μg/ml) for 2′-2 h. The levels of HO-1 and DNA repairsignaling proteins activation was measured by Western blot. As expected,camptothecin induced phosphorylation of H2AX in wild-type HEK293 cells,which was slightly enhanced in cells without HO-1 (FIG. 3A). Thesefindings correlated with phosphorylation of H2AX observed in the tissuesfrom Hmox1^(−/−) mice (FIG. 1).

The status of the major DNA repair kinases, ATR, and ATM and theirdownstream targets was evaluated in HEK293 cells treated withcamptothecin. Phosphorylation of ATR, ATM as well as p53, Chk2 and Brca1were relatively unchanged in stable LMP-infected HEK293 control cells ascompared to non-transfected naïve HEK293 cells (FIG. 3B). Knockdown ofHO-1 however resulted in a significant decrease in phosphorylation ofATM, Brca1, ATR and Chk2 (FIG. 3B). Importantly, there were nosignificant changes in phosphorylation of p53, suggesting that HO-1 isspecific and important in DNA repair rather than regulating apoptosis orcell cycle progression in the presence of a genotoxic stressor.

HEK293 cells treated with doxorubicin responded similarly with anincrease in phosphorylation of ATM, p53 and H2AX (FIG. 3C). HEK mirHO-1and control cells treated with doxorubicin (10 μg/ml) for 2′-2 h isshown. Knockdown of HO-1 under these conditions resulted in inhibitionin phosphorylated ATM and a delay in Brca1 activation. No significantchanges were observed in P-p53 or γ-H2AX in control ormirHO-1-transfected HEK293 cells in the absence of doxorubicin. Further,the effects of irradiation on DNA were investigated in HEK cells in thepresence and absence of HO-1 after irradiation (10 Gy). Thephosphorylation levels of ATM, Brca1, H2AX and p53 were significantlydecreased or delayed in mirHO-1 HEK cells compared to controls (FIG.3D). There were no significant changes in the total levels of major DNArepair complexes involved in HR-mediated DNA repair in mirHO-1 HEKcells. Akt is a loading control for the immunoblot for P-p53.

Example 3 HO-1/CO Facilitate HR Repair of Double Strand Breaks

Since Hmox1^(−/−) cells showed altered H2AXγ foci formation in responseto doxorubicin, and mirHO-1 HEK cells showed diminished activation ofmajor signaling cascades leading to DNA repair in response tocamptothecin, the role of HO-1 was assessed in homologousrecombination-mediated DNA repair, which is critical in DSB repairinduced by doxorubicin or camptothecin (during replication). To testthis, the U20S cell line containing the HR/SCR (homologousrecombination/sister chromatid recombination) reporter was used aspreviously described (12, 13). I-SceI endonuclease was used to introducea double strand break (DSB) within the reporter in U2OS cells. Theefficiency of homologous recombination induced by I-SceI correlated withgeneration of wild type GFP by gene conversion.

To test the role of HO-1 in DSB, HO-1 was transiently overexpressedtogether with the I-SceI plasmid in U2OS cells and tested the number ofcells positive for GFP 24 hours post-transfection (FIG. 4A). U20S-SCRreporter cells were co-transfected transiently with SceI and HO-1 orcontrol plasmid. Cells were transfected with HO-1 construct and theamount of GFP-positive cells was measured 48 hours after transfection. Asignificant 2-fold increase in GFP levels was observed in HO-1overexpressing cells as assessed by fluorescence microscopy and flowcytometry (FIGS. 4B-D) while control, nontransfected, as well ascontrol-vector-transfected cells showed a low frequency ofGFP-positivity indicating poor repair (FIGS. 4F-D). These data support arole for HO-1 in the induction of HR in response to DSB.

Many of the effects observed with HO-1 are mediated by one or more ofits enzymatic products biliverdin or CO. Therefore, the role ofbiliverdin and CO was tested separately in HR during DSB and DNA repair.U2OS-SCR reporter cells after transfection with SceI for 24 hours weretreated with CO 250 (ppm) for 24 hours. Biliverdin treatment had noeffect on the number of GFP-positive cells, however CO (250 ppm)significantly increased the number of GFP-positive cells (FIGS. 4E-G)suggesting that HO-1 acts in part via CO to regulate DNA repair pathwaysand facilitate HR in response to DNA damage. No effects of CO wereobserved on NHEJ-mediated DNA repair as measured by number ofXHATM-resistant colonies.

Example 4 DNA Repair Responses Modulated by CO/HO-1 are Dependent onATM/ATR Activity

Lysates of PC3 and HEK cells were treated with CO (250 ppm) for 2′ to 1h. CO induced rapid phosphorylation of Brca1 in HEK cells, as well asthe upstream kinases ATR and ATM (FIGS. 5A-5B), suggesting that COinduces DNA repair pathways in cells, which are constantly under risk ofoxidative DNA damage. Therefore, the two major DNA repair kinases, ATMand ATR, were examined to see if they are implicated in accelerated HRin the presence of CO or HO-1. The selective inhibitor of ATM and ATRkinases, CGK733, and the more selective inhibitor of ATM, KU55933, wasused to test this hypothesis. U20S-SCR reporter cells were transfectedwith SceI for 24 hours and treated with CGK733 (20 μM) or DMSO for 1hour prior treatment with CO (250 ppm) for 24 hours. U20S-SCR reportercells were co-transfected with SceI for 24 hours and KU55933 (20 μM) orDMSO for 1 hour prior treatment with CO (250 ppm) was applied forfollowing 24 hours. Blockade of ATM/ATR or ATM alone significantlydecreased CO-mediated HR (FIGS. 5C-E). U20S-SCR reporter cells wereco-transfected with SceI and HO-1 for 24 hours and CGK733 was appliedfor following 24 hours. Induction of HO-1 did not increase HR in thepresence of GK733 as otherwise observed in vehicle controls (FIG. 5F)strongly suggesting that both ATR and ATM are utilized by CO/HO-1 inpart to regulate HR and DNA repair following damage.

Example 5 CO Protects Against DNA Damage In Vivo in Response toChemotherapy or Radiation

Based on the effects observed in vitro, the salutary effects of CO weretested in an in vivo model of DNA damage in mice. Tissues were harvested14 days after treatment of nude mice with established PC3 tumors (2weeks) with CO (Control) or doxorubicin (8 mg/kg, twice per week,i.v.)±CO (daily, 1 hour, 250 ppm). Doxorubicin was employed to induceDNA damage in the tissues during chemotherapeutic treatment of tumorxenografts in nude mice. Doxorubicin led to substantial DNA damage inthe lung, liver, kidney and colon and to a lower extent in the spleen asdetermined by accumulation of H2AXγ foci and was associated withincreased expression of HO-1 (FIGS. 6A-E). CO significantly decreasedthe severity of DNA damage in all organs (FIGS. 6A-F).

To support the observations with chemical genotoxins, aradiation-induced model of DNA damage was also used. Endogenous HO-1 isinduced early in response to IR or doxorubicin and its expression issustained for 24-72 hours in the spleen, liver and kidney (FIGS. 7A-B).Kidneys and spleen were harvested after 2 hours after irradiation. Toevaluate the role of exogenous CO, mice were pretreated with CO for 1hour prior to either a lethal or sublethal dose of irradiation and thendaily for 1 hour. Spleens and intestines of mice were pretreated withAir or CO (250 ppm) 1 hour prior lethal dose of irradiation (10 Gy) andtreated daily with CO for 11 days. A chronic elevation of H2AXγ foci wasobserved in the spleen and intestine of irradiated mice (FIGS. 7C-D). Incontrast, treatment of mice with CO led to significant inhibition insustained H2AXγ foci formation that corresponded to a longer survivalrate with 80% of mice alive >10 days vs. 50% in air-treated animals(p=0.031*, CO versus Air, n=6/group). In contrast, there was a stronginduction of P-p53, P-Brca1, P-ATM and early induction of P-H2AX inperipheral blood mononuclear cells and bone marrow of irradiated animalstreated with CO for 1 hour as compared to air controls suggesting earlyresolution of DNA damage (FIGS. 7E-F). Tissues were harvested 2 hoursafter irradiation. To evaluate the role of H2AX signaling in CO effectson DNA repair, marginal bone marrow transplantation of BM cells fromH2ax^(−/−) and H2ax^(+/+) mice into wild type irradiated recipients wasused. Mice were treated with CO prior lethal dose of irradiation (10 Gy)and thereafter after receiving BM. Lack of H2AX in donor BM cellsreversed CO-mediated protection against irradiation-induced death,suggesting a critical role of the H2AX pathway in CO-mediated protection(FIG. 7G).

Example 6 Materials and Methods

Animals, Irradiation and CO Treatment

Male C57BL/6 mice 7-9 weeks of age were purchased from JacksonLaboratories (Bar Harbor, Me.). Mice were held under SPF conditions andthe experiments were approved by the IACUC at BIDMC. Mice wereirradiated with 10 Gy (lethal dose) or 5 Gy (sublethal dose) and theprotocol was approved by Radiation Safety Officer at BIDMC and theIACUC. Mice were exposed in plexiglass chamber to CO for 1 hour, 250 ppmprior irradiation and thereafter every day for 1 hour.

Marginal Bone Marrow Transplant

Mice were lethally irradiated and transplanted with 2.5 min cells i.v.immediately after irradiation. The amount of injected BM was sufficientfor recovery of 40% mice.

Nu/nu mice were purchased from Taconic (Hudson, N.Y.) at 7 weeks of age.Those mice were used in a complementary experiment of a subcutaneoustumor model of PC3 cells injected into the right flank of mice.Doxorubicin (8 mg/kg, Sigma) was given i.v. twice per week and mice wereexposed to CO daily (250 ppm for 1 hour). Tissues were harvested after 2weeks of treatment.

Cell Culture and Treatment

U203 HR/SCR cells were maintained in the DMEM medium (Gibco, Invitrogen)containing 10% FBS and antibiotics. pPHW1 cells (NHEJ model) weremaintained in the DMEM medium with 10% FBS and antibiotics. Mouse kidneyfibroblasts were obtained from adult Hmox1^(−/−) and Hmox1^(+/+) miceand culture between passages 4-7 in DMEM medium supplemented with 10%FBS and antibiotics. HEK293 were purchased from ATCC and were culturedfollowing manufacturer's protocol. For CO in vitro studies, cells wereexposed to 250 ppm CO, 5% CO₂ in 95% N₂ for 2 minutes to 24 hours. Cellswere irradiated with 10 Gy and harvested at different time points aftersingle exposure.

ShmirRNA Retroviral Mediated Transfections

MicroRNA-adapted shRNA (shRNAmir) for human HO-1 was purchased from OpenBiosystems (AL, USA). shRNAmir-HO-1 was subcloned to MSCV-LTRmiR30-PIG(LMP) vector (Open Biosystems) with XhoI and EcoRI restriction enzymes.The retrovirus for LMP-shRNAHO-1 and control vector were produced andused for transduction of HEK-293 cells. Stable clones were generated byselection with 5 μg/ml puromycin (Sigma) for 2-4 weeks.

HR/SCR Reporter Assay, Flow Cytometry and Fluorescence Microscopy

HR/SCR reporter U20S cells were used as previously described. Briefly,GFP+ cells were analyzed 2 days postransfection by flow cytometry(FACScan, BD Biosciences) and fluorescence microscopy. Images ofGFP-positive cells were captured of randomized fields using a ZeissApotome fluorescent microscope.

Statistical Analyses

The significance of differences was determined using analysis ofvariance (ANOVA) or Student t-test (SPSS Inc, Chicago, Ill.) withsignificance accepted at p<0.05. For survival analysis Log Rank MantelCox test was applied.

Example 7 Carbon Monoxide in Aging and Senescence

This example demonstrates that heme oxygenase-1 and CORM inhibit thesenescence-associated β-galactosidase staining in a zebrafish embryomodel of aging.

A model of senescence in vivo in the zebrafish embryos was employed aspreviously described (Koshimizu et al., 2011, Plos One, 6:e17688).Embryos were either transfected with a morpholino to inhibit HO-1expression or a control vector. In a separate treatment group, embryoswere transfected with the HO-1 morpholino and treated with aCO-releasing molecule (50 μM; Sigma-Aldrich). Results show that embryoslacking HO-1 have a greater β-gal activity indicating greater or morerapid senescence and that CO administration can reverse this to a normalsenescence/aging level of positive β-gal staining, trending towardsslowing of senescence. Knockdown of HO-1 with morpholinos against HO-1in zebrafish embryos resulted in induction of senescence-associatedβ-galactosidase (SA-β-gal) activity as compared to control morpholinosor non-injected larvae (FIG. 8). A slowing of senescence of zebrafishembryos was observed with application of CORM after injection withmorpholinos against HO-1 (FIG. 8). These data suggest that agingassociated senescence that is accelerated in the absence of HO-1 can bereversed by application of CO.

In another set of experiments performed in mice, mice were exposed to COfor 1 hour and harvested multiple tissues at 24 hours and found thatanimals treated with CO had increased expression of the anti-aging genesSIRT1 (sirtuin; FIG. 9A) and TERT (telomerase reverse transcriptase;FIG. 9B) in the lung. This is important given that the lung has such ahigh rate of cellular turnover. The increase in expression of thesegenes is associated with decreased senescence and extension of lifespan.

Example 8 CO in DNA Repair

This example demonstrates that exposure to CO increases expression oftelomerase, topoisomerase, and p16 to decrease DNA damage.

Male C57B1/6 mice were exposed to inhaled carbon monoxide at 250 partsper million(ppm) for 1 hour. Mice were immediately removed from theexposure chamber and the lungs and intestines were harvested andprocessed for RNA. Real time PCR for telomerase in lungs, andtopoisomerase (responsible for unwinding of DNA) and p16 (cell cyclesuppressor) in intestines was performed, and expression was compared toair-exposed controls (FIG. 10). Results represent mean±SD of at least 3mice/group, *p<0.05.

OTHER EMBODIMENTS

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1.-13. (canceled)
 14. A method of administering a genotoxic treatment toa patient, the method comprising: (a) administering the genotoxictreatment to the patient; and (b) before, during, or after step (a),administering to the patient a pharmaceutical composition comprisingcarbon monoxide in an amount effective to protect cells of the patient,wherein the genotoxic treatment is radiotherapy or hyperthermia therapy.15. The method of claim 14, wherein the pharmaceutical composition isadministered before step (a).
 16. The method of claim 14, wherein thepharmaceutical composition is administered during step (a).
 17. Themethod of claim 14, wherein the pharmaceutical composition isadministered after step (a).
 18. The method of claim 14, wherein thepharmaceutical composition is administered before, during, and afterstep (a).
 19. The method of claim 14, wherein the genotoxic treatment isradiotherapy.
 20. The method of claim 14, wherein the genotoxictreatment is hyperthermia therapy.
 21. The method of claim 14, whereinthe pharmaceutical composition is in gaseous form and is administered tothe patient by inhalation.
 22. The method of claim 14, wherein thepharmaceutical composition is in liquid form and is administered to thepatient orally.
 23. The method of claim 14, wherein the pharmaceuticalcomposition is administered directly to the abdominal cavity of thepatient.
 24. The method of claim 14, wherein the pharmaceuticalcomposition comprises a carbon monoxide-releasing compound.
 25. Themethod of claim 14, wherein the pharmaceutical composition isadministered by an artificial lung.
 26. The method of claim 14, whereinthe pharmaceutical composition is administered by an extracorporealmembrane gas exchange device.
 27. A method of ameliorating age-relateddamage to DNA in a patient, the method comprising administering to thepatient a pharmaceutical composition comprising an amount of carbonmonoxide effective to ameliorate age-related damage to DNA in thepatient.
 28. The method of claim 27, wherein the pharmaceuticalcomposition is in gaseous form and is administered to the patient byinhalation.
 29. The method of claim 27, wherein the pharmaceuticalcomposition is in liquid form and is administered to the patient orally.30. The method of claim 27, wherein the pharmaceutical composition isadministered directly to the abdominal cavity of the patient.
 31. Themethod of claim 27, wherein the pharmaceutical composition comprises acarbon monoxide-releasing compound.
 32. The method of claim 27, whereinthe pharmaceutical composition is administered by an artificial lung.33. The method of claim 27, wherein the pharmaceutical composition isadministered by an extracorporeal membrane gas exchange device.